Primary pulmonary diffuse large B-cell lymphoma associated with feline leukaemia virus infection in a young cat

Case description

A 4-year-old castrated male domestic shorthair cat with a continuous cough was brought to a private veterinary clinic for detailed examination. Physical examination, complete blood count and routine serum biochemical profile indicated no abnormalities. The cat was annually vaccinated with a triple vaccine against the feline herpesvirus, feline calicivirus and feline parvovirus but not against the feline leukaemia virus (FeLV). Serological analysis for the FeLV antigen and for the antibody against the feline immunodeficiency virus (FIV) yielded positive results. Radiography of the thoracic cavity revealed a severely radiopaque region in the caudal lobe of the left lung. The cat was prescribed 7 mg/ml enrofloxacin (Baytril; Bayer) by a private veterinarian, and its clinical signs improved following the drug’s administration.

At 48 days after the initial visit, the cat was brought to the Veterinary Medical Centre, Nippon Veterinary and Life Science University, with a recurrent cough for further detailed examination. Analysis for FeLV antigen (but not for the anti-FIV antibody) yielded a positive result on the same day; however, regular virus tests were not performed, at the request of the owner. At radiography, a radiopaque region was noted; bronchopneumonia was suspected in the caudal lobe of the left lung and a nodule measuring approximately 11 × 7 mm was detected in the caudal lobe of the right lung. Doxycycline hydrochloride hydrate (7 mg/kg q12h for 13 days and then q24h for 66 days [vibramycin; Pfizer]) was administered to treat bronchopneumonia. No abnormalities in other organs were observed on thoracic radiography and abdominal ultrasonography; haematological and serum biochemical parameters were within normal limits.

At 90 days after the initial visit to the private veterinary clinic, the nodule in the caudal lobe of the right lung showed an increase in size to approximately 16 × 14 × 12 mm vs the size detected on radiography at 48 days; CT then showed an increase in the size of the mass in the caudal lobe of the right lung to 27 × 23 × 18 mm (Figure 1).

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Figure 1

CT of the thoracic cavity revealed a mass measuring 27 × 23 cm in the caudal lobe of the right lung

At 108 days after the initial visit, and a radiopaque region in the accessory lobe was revealed. Fine-needle aspiration (FNA) was not performed because the mass was located far from the thoracic margin. None of the sternal, cranial mediastinal, tracheobronchial nor abdominal lymph nodes was enlarged, and abdominal organs, including the liver, kidneys, spleen and gastrointestinal tract, appeared normal on CT examination of the whole body under contrast medium administration. On the same day, pulmonary lobectomy of the caudal lobe of the right lung and accessary lobe was performed via an incision made at the fifth intercostal muscle. General anaesthesia was induced with 1% propofol (Propoflol; Fresenius Kabi) intravenous injection until the desired effect was obtained. As soon as jaw relaxation was achieved, endotracheal intubation was performed. Anaesthesia was maintained with 100% oxygen and isoflurane (isoflurane inhalation solution; Pfizer), ranging from 1.0% to 1.9%. Rocuronium bromide (Eslax; MSD) was administered at a dose of 0.5 mg/kg intravenously for the induction of muscle relaxation. The cat was infused with 2–8 µg/kg/min fentanyl citrate (fentanyl injection; Terumo) during the operation, as well as at 10 h postoperatively for pain management. Cefazolin (25 mg/kg cefazolin sodium; Nipro) was administered every 2 h to prevent infection perioperatively and every 12 h postoperatively. The resected tissues were submitted to the Department of Veterinary Pathology, Nippon Veterinary and Life Science University.

Grossly, the mass located in the posterior lobe of the right lung had a white surface and was firm (Figure 2). A nodule-like lesion in the accessory lobe showed a pale-reddish colour. The resected pulmonary lobes were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 µm) were stained with haematoxylin and eosin, Gram, periodic acid–Schiff (PAS) and Grocott stains. Microscopically, the mass was non-encapsulated with an unclear margin with the surrounding tissues. It comprised large round or polygonal neoplastic cells arranged in a diffuse pattern. The neoplastic cells displayed large, round, hypochromatic nuclei with scant pale eosinophilic cytoplasm (Figure 3). The frequency of mitosis was ⩾2 per high-power field (× 400). No intravascular embolisation by neoplastic cells was identified. Immunohistochemical (IHC) analysis was performed with the resected caudal right lung mass using labelled streptavidin–biotin and primary antibodies specific for CD3, CD20, CD204, E-cadherin, the ionised calcium-binding adaptor molecule-1 (Iba-1), granzyme B, FeLV p27 and FeLV glycoprotein (gp) 70 (Table 1), to distinguish between T-cell lymphoma, B-cell lymphoma and pulmonary Langerhans cell histiocytosis. A combination of CD20 and FeLV p27 antibodies was also used to perform double-labelled immunofluorescence. Neoplastic cells were diffusely positive for CD20 but negative for CD3, CD204, Iba-1, granzyme B and E-cadherin. The cytoplasm of neoplastic cells was positive for FeLV p27 and gp70. Neoplastic cells coexpressed CD20 and FeLV p27 (Figure 4). T lymphocytes and macrophages were often observed in the peripheral area of neoplastic tissue.

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Figure 2

Macroscopical appearance of the pulmonary mass removed from the caudal right lung lobe. Scale bar = 2 cm

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Figure 3

The mass comprised round-to-polygonal neoplastic cells arranged in a diffuse pattern. Haematoxylin and eosin. scale bar = 50 µm

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Table 1

Primary antibodies used in the immunohistochemistry analysis

Antibody	Host (clone)	Dilution	Antigen retrieval	Source
CD3	Mouse (F7.2.38)	1:300	121°C for 20 mins in citrate buffer, pH 6.0	Dako
CD20	Mouse (N/A)	1:200	121°C for 20 mins in citrate buffer, pH 6.0	Biocare Medical
CD204	Mouse (SRA-E5)	1:100	Microwave for 10 mins in citrate buffer, pH 2.0	TransGenic
E-cadherin	Mouse (NCH-38)	1:100	121°C for 10 mins in Tris-EDTA buffer, pH 9.0	Dako
Granzyme B	Mouse (GrB-7)	1:50	121°C for 20 mins in citrate buffer, pH 6.0	Nichirei Biosciences
FeLV p27	Mouse (PFI2J-10A)	1:100	121°C for 20 mins in citrate buffer, pH 6.0	Custom Monoclonals International
FeLV gp70	Mouse (C11D8)	1:200	121°C for 20 mins in citrate buffer, pH 6.0	Bio-Rad Laboratories
Iba-1	Rabbit	1:1,000	121°C for 20 mins in citrate buffer, pH 6.0	Wako

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Figure 4

Double immunofluorescence staining showing coexpression of CD20 (green) and feline leukaemia virus p27 (red) in neoplastic cells. Scale bar = 50 µm

Based on the morphological and IHC findings, the neoplasm was diagnosed to be diffuse large B-cell lymphoma, according to the World Health Organization (WHO) classification of malignant lymphoma in domestic animals. ¹ Chronic bronchopneumonia was observed in the accessory lobe; however, no lymphoma or bacterial or fungal infections were observed on histopathological examination using special stains, including Gram, PAS and Grocott. Additional chemotherapy according to the cyclophosphamide, doxorubicin/hydroxydaunorubicin, vincristine/oncovin and prednisolone (CHOP) protocol ² was administered following surgical excision, in addition to doxycycline hydrochloride hydrate (7 mg/kg q24h). In a detailed examination performed 196 days after surgical resection, abdominal ultrasonography showed thickening of the jejunum, and a subsequent FNA under ultrasonography detected a high-grade lymphoma arising from the jejunum (Figure 5). The cat showed no associated clinical signs such as weight loss, vomiting or diarrhoea, and tumour lesions in the lung or other haematopoietic organs were not seen. Bronchopneumonia was immediately cured after doxycycline hydrochloride hydrate administration, although the cat showed slight respiratory signs after lobectomy. At 224 days after surgical resection, the cat died with black defecation suggestive of intestinal haemorrhage a few days previously.

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Figure 5

Fine-needle aspirate cytology of the jejunum revealed large round cells with rounded nuclei and small amounts of basophilic cytoplasm, consistent with high-grade lymphoma. Wright–Giemsa stain, scale bar = 20 µm

Discussion

Primary pulmonary neoplasms, such has adenocarcinoma, bronchioloalveolar carcinoma, adenosquamous carcinoma, carcinosarcoma or pulmonary Langerhans cell histiocytosis, are uncommon in cats.^2–7 However, lymphomas are common neoplasms in cats. ⁸ Based on their anatomical origin, feline lymphomas are classified into five types: multicentric, alimentary, mediastinal, cutaneous and extranodal. ⁸ Cats predominantly present with alimentary and mediastinal lymphoma types. ⁸ Lymphomas occurring in the lungs are classified into primary neoplasms, and secondary involvement of those arising from the other thoracic organs (such as the thymus, mediastinal, sternal or tracheobronchial lymph nodes) or from extrathoracic organs (such as the liver, kidneys and spleen).^9,10 However, to our knowledge, pulmonary involvement of feline lymphoma has not previously been reported in cats. Based on the histological appearance, the most distinct lung neoplasm is Langerhans cell histiocytosis. Pulmonary Langerhans cell histiocytosis originates from dendritic cells that express CD18, E-cadherin and Iba-1 but not CD204; initially, it results in the formation of a nodular lesion surrounding the bronchi before spreading throughout the lung.^5,7 Pulmonary Langerhans cell histiocytosis can be excluded based on the aforementioned IHC features.

The WHO classification of tumours of the lung in humans subclassifies lymphohistiocytic tumours to include extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue, diffuse large B-cell lymphoma, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, pulmonary Langerhans cell histiocytosis and Erdheim–Chester disease. ¹¹ In humans, the occurrence of primary pulmonary lymphoma is extremely rare, representing <1% of all lymphomas, and it is defined as clonal proliferation in the parenchyma of the lung in a patient with no detectable extrapulmonary involvement at diagnosis or during the subsequent 3 months.^12,13 In cats, primary pulmonary lymphoma has not yet been reported, although secondary involvement of lymphoma in the lung has been reported.^9,10,14 Pulmonary involvement of lymphoma is observed in 12% of the cases (16/125 on necropsy). It forms macroscopic masses and nodules, and shows histologically predominant peribronchial invasion, followed by pleural, interstitial, nodular and alveolar patterns. ¹⁰ Although no clinical signs were noted for almost 7 months after surgery and chemotherapy based on the CHOP protocol, abdominal ultrasonography and FNA examination detected high-grade lymphoma in the jejunum. No gastrointestinal signs or abnormal findings in the gastrointestinal tract, including the jejunum, were detected on abdominal ultrasonography after lobectomy. Furthermore, lymphoma occurring in the right caudal lung lobe was considered a primary pulmonary neoplasia as jejunal lymphoma was not detected until 196 days after surgical resection.

FeLV causes immune dysfunction by suppressing immunoglobulin production and neutrophil function in infected cats. ¹⁵ Furthermore, FeLV is involved in the development of various tumours, including haematopoietic tumours such as lymphoma and leukaemia, and non-haematopoietic tumours such as osteochondroma. Overall, 15% or 25% of infected cats develop cancers such as lymphoma.^15,16 The most important mechanism by which as FeLV causes tumours is that the FeLV genome is integrated near oncogenes such c-myc and bmi-1, causing activation and overexpression of these genes. ¹⁷ . FeLV-induced lymphomas derived from both T and B lymphocytes occur as several types, including multicentric, mediastinal, alimentary and renal types, as well as upper respiratory type, which induce the development of diffuse B-cell lymphoma.^17–21 FeLV infection in feline lymphoma can be demonstrated by IHC staining of FeLV capsid p27 and envelope gp70.^10,20,22,23 On IHC, FeLV-positive cats are immunohistochemically regarded as productively FeLV-infected cats. ²² Moreover, 87% (34/39 cases) of lymphomas occurring in the upper airways such as the nasal cavity and nasopharynx have been found to be of the B-cell type, and in 62% of these cases (n = 21/34) FeLV p27 and/or gp70 were detected on IHC examination. ²⁰ FeLV may be involved in the development of lymphoma in the upper respiratory tract in cats, although the association between serum FeLV test- and IHC-positive cases has not been evaluated owing to the small number cases in which serum FeLV tests have been performed. ²⁰ FeLV viral RNA can also be detected by in situ hybridisation in case of B-cell lymphoma of the spinal cord. ²⁴ In the present case, FeLV antigen was detected by both serum tests and IHC staining, suggesting that tumourigenesis in this case was related to the effects of FeLV infection on immune surveillance.

Conclusions

To the best of our knowledge, this is the first report of a primary pulmonary diffuse B-cell lymphoma case associated with FeLV infection in a young cat.